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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
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  • C12N15/09—Recombinant DNA-technology
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering N.A.
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/315—Phosphorothioates
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate

Definitions

• the present invention relates to a nucleic acid complex, a pharmaceutical composition comprising the nucleic acid complex, and others.
• nucleic acid drugs are, for example, antisense drugs, decoy nucleic acids, ribozymes, aptamers, siRNA, miRNA, and messenger RNA (mRNA).
• nucleic acid drugs which act on mRNA, are expected to be promising for clinical application to diseases previously considered to be intractable because of their high versatility that allows all kinds of genes in cells to be regulated.
• nucleic acid drugs are expected to be promising as next-generation drugs following small molecules and antibody drugs.
• nucleic acid drugs suffer from a problem of difficulty in delivering into cells, which are sites of action for nucleic acid drugs, because of, for example, the relatively large size of nucleic acid drugs, significantly low cell membrane permeability due to negative charges in the phosphate backbone, and decomposition of siRNA by nucleases in the blood (Non Patent Literature 1).
• nucleic acid complexes conjuggated nucleic acids formed of a target ligand and a nucleic acid have been reported as one of delivery means.
• the target ligand include a form that binds to a receptor expressed on cell surfaces.
• GalNAc N-acetyl-D-galactosamine.
• ASGPR asialoglycoprotein receptor
• a mannose conjugate and a mannosylated nucleic acid complex have been reported as drug carriers for delivery to the mannose receptor (CD206), which is highly expressed on immunocytes such as macrophages and dendritic cells (Patent Literature 4). Further, it has been reported that a nucleic acid drug is modified with a liposoluble compound such as cholesterol and fatty acid to enhance the affinity with lipoproteins and the like in the plasma, thereby achieving delivemy to cells expressing the corresponding lipoprotein receptors (LDL receptor, HDL receptor, scavenger receptor SRB1) Non Patent Literature 3).
• a liposoluble compound such as cholesterol and fatty acid
• Patent Literature 1 WO 2009/073809
• Patent Literature 2 WO 2014/179620
• Patent Literature 3 WO 2016/100401
• Patent Literature 4 WO 2018/004004
• Non Patent Literature 1 Nature Reviews Drug Discovery. 8, 129-138 (2009).
• Non Patent Literature 2 J. Am. Chem. Soc. 2014, 136, 16958-16961.
• Non Patent Literature 3 Nucleic Acids Research, 2019, Vol. 47, No. 3, 1082-1096.
• the present inventors diligently studied to solve the problem, and eventually found a nucleic acid complex that is taken up in a cell-selective manner.
• the present invention relates to [1] to [27] in the following.
• nucleic acid complex according to any one of [1] to [20] or the pharmaceutical composition according to [21] to the subject.
• the nucleic acid complex according to the present invention is selectively taken up by cells expressing the mannose receptor or ASGPR.
• the nucleic acid complex according to the present invention has a potential to cure various associated diseases through administration of a pharmaceutical composition convising the nucleic acid complex according to the present invention to mammals (including humans).
• the structural formula occasionally represents a certain isomer for convenience, but the scope of the compound is not limited to the illustration of the thrmula for convenience, and includes all the structurally acceptable isomers including geometric isomers, optical isomers, rotamers, stereoisomers, and tautomers, and isomer mixtures, and the compound may be any one isomer or a mixture containing the isomers at any ratio. Accordingly, while optical isomers and a racemate may exist for a compound in the present specification, for example, the compound is not limited to any of them and may be the racemate, or any of the optically active forms, or a mixture containing the optically active forms at any ratio.
• crystalline polymorphs may exist :for a compound in the present specification, the compound is not limited to any ofthem, similarly, and May be a single substance of any of the crystal forms or a mixture thereof, and the scope of a compound in the present specification includes its amorphous forms, and the scope of a compound in the present specification encompasses its anhydride and solvates (in particular, hydrates).
• the scope of a compound in the present specification also includes isotope-labeled forms of the compound.
• An isotope-labeled compound is the same as the original compound except that one or more atoms have been replaced with atoms each having an atomic mass or mass number differing from the atomic mass or mass number typically found in the natural world.
• Isotopes that can be incorporated in a compound in the present specification are isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, phosphorus, sulfur, iodine, and chlorine, and examples thereof include 2 H, 3 H, 11 C, 14 C, 15 N, 18 O, 18 F, and 35 S.
• the “pharmaceutically acceptable salt” in the present specification is not limited as long as the pharmaceutically acceptable salt is formed by salt formation with a compound, and specific examples thereof include acid addition salts, metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts.
• acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate, and organic acid salts such as acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, p-toluenesulfonate, and benzenesulfonate.
• inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate
• organic acid salts such as acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, p-toluenesulfonate, and benzenesulfonate.
• metal salts include alkali metal salts such as sodium salt and. potassium salt, alkali earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt.
• ammonium salts include salts of ammonium, tetramethylammonium, and the like.
• organic amine addition salts include addition salts of morpholine, piperidine, and the like.
• amino acid addition salts include addition salts of lysine, glycine, phenylalanine, aspartic acid, glutamic acid, and the like.
• a compound in the present specification is obtained as a free form, the compound can be converted into a state in which a salt that the compound may be forming or a hydrate thereof in accordance with a conventional method.
• a compound in the present specification is obtained as a salt or hydrate
• the salt or hydrate can be converted into the free form in accordance with a conventional method.
• Various isomers e.g., geometric isomers, optical isomers, rotamers, stereoisomers, tautomers
• a common separation means such as recrystallization, diastereomeric salt formation, enzymatic resolution, and various kinds of chromatography (e.g., thin-layer chromatography, column chromatography, gas chromatography, high-performance liquid chromatography).
• the pharmaceutical composition according to the present invention can be produced by sufficiently mixing a pharmaceutically acceptable excipient with the nucleic acid complex or a pharmaceutically acceptable salt thereof.
• the pharmaceutical composition according to the present invention can be produced in accordance with a known method such as a method described in General Rules for Preparations, The Japanese Pharmacopeia, Seventeenth Edition.
• composition according to the present invention can be administered to a patient in a proper manner according to the dosage form.
• the dose of the nucleic acid complex according to the present invention depends on the degree of the symptoms, the age, sex, and body weight, the mode of administration and the type of the salt, the specific type of the disease, and so on, and a daily dose of approximately 30 ⁇ g to 10 g, preferably 100 ⁇ g to 1 g, in the case of oral administration, or a daily dose of approximately 1 ⁇ g to 1 g, preferably 100 ⁇ g to 300 mg, in the case of administration by injection, in terms of oligonucleotide is typically administered to an adult at once, or separately in several portions.
• the nucleic acid complex according to the present invention is a nucleic acid complex comprising a sugar ligand bound to an oligonucleotide via a linker, wherein the sugar ligand has O-, N-, or C-linked, preferably O-linked mannose or GalNAc. That is, the nucleic acid complex according to the present invention has the structure (sugar ligand)-(linker)-(oligonucleotide).
• the nucleic acid complex comprises two or more sugar moieties, preferably three or four sugar moieties.
• the nucleic acid complex comprises at least three mannose moieties or at least three GalNAc moieties, and the nucleic acid complex can he delivered to macrophages or liver parenchymal cells as the target.
• the mannose receptor is highly expressed on cells highly expressing CD206, for example, specific cells including macrophages and dendritic cells.
• Mannose conjugates and mannosylated drug carriers exhibit high binding affinity with CD206, and are successfully used to deliver drug molecules such as oligonucleotides to cells including macrophages and dendritic cells.
• ASGPR is highly expressed on specific cells including liver parenchymal cells.
• GalNAc conjugates and GalNAc-conjugated drug carriers exhibit high binding affinity with ASGPR, and are successfully used to deliver drug molecules such as oligonucleotides to cells including liver parenchymal cells.
• sugar ligand refers to a group derived from a sugar capable of binding to a receptor expressed on target cells
• one of preferred modes of the sugar ligand in the nucleic acid complex according to the present invention may be, for example, the following structure.
• Each wavy line indicates a bond to the linker.
• any oligonucleotide known to be applicable as a nucleic acid drug can be used.
• the teen nucleic acid drug refers to nucleotides for use as an antisense drug, a decoy nucleic acid, a ribozyme, siRNA, miRNA, anti-miRNA, mRNA, or the like.
• the oligonucleotide may be a single-stranded or double-stranded oligonucleotide.
• the linker and the oligonucleotide in the nucleic acid complex according to the present invention may be bound together, for example, at a nucleotide of the oligonucleotide, and are bound together, for example, at the 3′ end or 5′ end of the oligonucleotide. If the oligonucleotide is double-stranded, it is preferable that the linker be bound to the 3′ end or 5′ end of the sense strand constituting the double-stranded nucleic acid; however, the binding is not limited to the mentioned one.
• the number of linkers to which the oligonucleotide in the nucleic acid complex is bound is not limited to one, and may be two or more.
• inclusion of a specific base in an overhang or inclusion of a modified nucleotide or nucleotide substitute in a single-stranded overhang is acceptable for enhancing the stability.
• the oligonucleotide constituting the nucleic acid complex according to the present invention may be in any shape as long as the oligonucleotide has an ability to regulate expression of a target gene when being introduced into mammalian cells, and single-stranded oligonucleotide or double-stranded oligonucleotide is preferably used.
• the oligonucleotide may be any molecule as long as the oligonucleotide is a polymer of nucleotides or molecules having functions comparable to those of nucleotide, and examples thereof include DNA, which is a polymer of deoxyribonucleotides, RNA, which is a polymer of ribonucleotides, and chimeric nucleic acid, which is a polymer of DNA and RNA.
• the DNA, RNA, and chimeric nucleic acid may be each a nucleotide polymer in which at least one nucleotide such as deoxyribonucleotide and ribonucleotide is substituted with a molecule having functions comparable to those of nucleotide.
• Uracil (U) in RNA is uniquely interpreted as thymine (T) in DNA.
• nucleotide derivatives obtained by modifying nucleotide examples include nucleotide derivatives obtained by modifying nucleotide, and a molecule or the like obtained by modifying deoxyribonucleotide or ribonucleotide, for example, to enhance or stabilize the nuclease resistance, increase the affinity with the complementary nucleic acid, or increase the cell permeability as compared with DNA and RNA, or for visualization, is preferably used.
• nucleotide derivatives include nucleotide in which at least one of the sugar moieties, the phosphodiester bond, and the base has been modified, such as nucleotide with the sugar moiety modified, nucleotide with the phosphodiester bond modified, and nucleotide with the base modified.
• the nucleotide with the sugar moiety modified may be any nucleotide as long as the nucleotide is modified or substituted with any substituent or substituted with any atom partially or wholly in the chemical structure of the sugar of the nucleotide, and 2′-modified nucleotide is preferably used.
• Examples of the 2′-modified nucleotide include 2′-modified nucleotide in which the 2′-OH group of the ribose is substituted with a substituent selected from the group consisting of OR, R, R′OR, SH, SR, NH 2 , NHR, NR 2 , N 3 , CN, F, Cl, Br, and I (R is an alkyl or aryl, preferably an alkyl having one to six carbon atoms, and is an alkylene, preferably an alkylene having one to six carbon atoms), and preferred examples of the 2′-modification include substitution with F, substitution with a methoxy group, and substitution wth. an ethoxy group. Also acceptable is 2′-modified nucleotide in which the oxygen atom at the 2′ position and the carbon at the 4′ position in the ribose are crosslinked via methylene, that is, locked nucleic acid.
• a substituent selected from the group consisting of OR, R,
• the nucleotide with the phosphodiester bond modified may be any nucleotide as long as the nucleotide is modified or substituted with any substituent or substituted with any atom partially or wholly in the chemical structure of the phosphodiester bond of the nucleotide, and examples thereof include nucleotide in which the phosphodiester bond is substituted with a phosphorothioate bond, nucleotide in which the phosphodiester bond is substituted with a phosphorodithioate bond, nucleotide in which the phosphodiester bond is substituted with an alkylphosphonate bond, and nucleotide in which the phosphodiester bond is substituted with a phosphoramidate bond, and a preferred example is nucleotide in which the phosphodiester bond is substituted with a phosphorothioate bond.
• oligonucleotide encompasses oligonucleotide which some or all of the atoms in the molecule are substituted with an atom of different mass number (isotope).
• the oligonucleotide in the nucleic acid complex according to the present invention regulates expression of a target gene in cells.
• the oligonucleotide in the nucleic acid complex of the present invention binds to a ligand via a linker (also referred to as a linker-ligand).
• linker in the present invention, any linker that can be used for nucleic acid complexes can be used.
• any of the structures disclosed in WO 2009/073809, WO 2013/075035, and WO 2015/105083 can be employed as the linker structure.
• the linker comprises at least one cleavable linking group.
• cleavable linking group refers to a group that is sufficiently stable outside of cells, but is cleaved upon the entry to a target cell and releases the moiety to which the linker is bound.
• a phosphate-based cleavable linking group is cleaved by an agent that decomposes or hydrolyzes a phosphate group.
• An example in which a phosphate group is cleaved in cells is an enzyme such as phosphatase.
• a preferred embodiment of the phosphate-based linking group is —O—P(O)(OH)—O— or O—P(S)(OH)—O—.
• linker is any one of the following structures, wherein X and Y, independently at each occurrence, X represents CH 2 or O, and Y represents a sugar ligand, respectively, Z represents a group containing oligonucleotide, and n is an integer of 1 to 8:
• the nucleic acid complex according to the present invention can be produced with any method described in production examples below, and the effects of the compound can be confirmed with methods described in Test Examples below. However, those are only examples, and the present invention is not limited to specific examples below in any case, and may be modified without departing from the scope of the present invention. Examples in the present invention can be produced by using synthetic chemistry techniques known to those skilled in the art.
• Root temperature in Examples and production examples below refers to approximately 10° C. to approximately 35° C. in normal cases. Unless otherwise specified, % indicates percent by weight or volume.
• silica gel column chromatography Silica Gel 60 (70 to 230 mesh, or 230 to 400 mesh ASTM) manufactured by Merck KGaA, PSQ60B manufactured by FUJI SILYSIA CHEMICAL LTD., or a prepacked column (column: Hi-Flash (TM) Column (Siticagel) manufactured by YAMAZEN CORPORATION, or a Biotage (TM) SNAP Ultra Silica Cartridge manufactured by Biotage) was used as the silica gel.
• TM Hi-Flash
• TM Biotage
• production can be performed with methods known to those skilled in the art.
• reaction mixture was diluted with DCM and filtered, and the filtrate was then washed with saturated aqueous sodium hydrogen carbonate solution, water, and saline, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure.
• the residue was purified by silica gel column chromatography (50:1 to 20:1 petroleum ether/EtOAc) to afford compound 4 (7.50 g).
• 6-azidehexanoic acid compound 7 (118 mg, 0.75 mmol), DIPEA (258 mg, 2.00 mmol), HBTU (270 mg, 712 ⁇ mol), and DMF (3 mL) were added, and the resultant was stirred at 25° for 1 hour.
• DIPEA 258 mg, 2.00 mmol
• HBTU 270 mg, 712 ⁇ mol
• DMF 3 mL
• Synthesis of SEQ-1 and SEQ-2 was outsourced to Gene Design Inc, Sodium tetraborate butler at pH 8.5 (final concentration: 40 mM) was added to SEQ-2 (1.3 ⁇ mol), DBCO-NHS ester (CAS No. 1353016-71-3, 60 ⁇ mol) dissolved in DMSO was added thereto, and the resultant was stirred at room temperature for 15 minutes. After adding water to the reaction mixture, gel filtration purification was performed with a PD-10 column (GE Healthcare). Further, purification and concentration were performed with an Amicon Ultra 3K (Millipore) to afford a crude product (SEQ-3).
• sequence of-the nucleic acid (5′-3′) used in Examples shown here is A(F) ⁇ circumflex over ( ) ⁇ G(M) ⁇ circumflex over ( ) ⁇ G(F)A(M)C(F)U(M)G(F)G(M)U(F)C(M)U(F)U(M)C(F)U (M)A(F)U(M)A(F)U(M) ⁇ circumflex over ( ) ⁇ C(F) ⁇ circumflex over ( ) ⁇ U(M), wherein capital alphabets represent RNA, (M) indicates 2′-O-methylated RNA, (F) indicates 2′-fluoroRNA, and ⁇ circumflex over ( ) ⁇ indicates phosphorothioate linkage.
• the cyanine dye Cy3 (excitation wavelength: 555 nm, fluorescence wavelength: 570 nm), which is a fluorescent dye, is bound to the 5′ end of each oligonucleotide.
• SEQ-1, 2 and the nucleic acid complexes synthesized in Examples shown here were subjected to measurement of molecul ar weight by MALDI-TOF-MS, and Table 1 shows the results.
• the nucleic acid complexes synthesized in Examples were each introduced into human CD206-expressing Lenti-X 293T cells (Clontech Laboratories, Inc.) with a method shown below, and the uptakes of them were evaluated.
• Human CD206-expressing Lenti-X 293T cells (Clontech Laboratories, Inc.) were seeded in a 96-well PDL coating plate (Corning Incorporated) at 2 ⁇ 10 4 cells/100 ⁇ L/well, and cultured in a 5% CO 2 incubator at 37° C. for 2 days.
• SEQ-1 or any one of the nucleic acid complexes synthesized was added to reach a final concentration of 100 nmol/L, and incubation was performed in a 5% CO 2 incubator at 37° C. for 2 hours. Thereto, 4% paraformaldehyde/PBS (Wako Pure Chemical Industries, Ltd.) containing 10 ⁇ g/mL Hoechst (Lite Technologies) was added, and the cells were fixed at normal temperature for 30 minutes, and washed four times with PBS. Fluorescence imaging analysis was performed with an IN Cell Analyzer 2200 (GE Healthcare), wherein Cy3 fluorescence intensity in each well was corrected with the number of nuclei, and the average Cy3 fluorescence intensity per cell was calculated.
• IN Cell Analyzer 2200 GE Healthcare
• the fluorescence intensity in the well with addition of SEQ-1 which is a nucleic acid without modification with a sugar ligand, was defined as 1, and fluorescence intensity in each well with addition of any one of the nucleic acid complexes was calculated as a relative value.
• Table 2 shows the results. It was revealed from the results that, as compared with SEQ-1, which contained no sugar ligand, nucleic acid complex 6, which had GalNAc, was not effectively taken up by CD206-expressing cells, whereas nucleic acid complexes 1 to 5, nucleic acid complexes having mannose, were effectively taken up by CD206-expressing cells.
• the nucleic acid complexes synthesized in Examples were each introduced into human ASGR1-expressing Lenti-X293T cells (Clontech Laboratories, Inc.) with a method shown below, and the uptake activity for them was evaluated.
• Human ASGR1-expressing Lenti-X293T cells (Clontech Laboratories, Inc.) were seeded in a 96-well PDL-coating plate (Corning Incorporated) at 2 ⁇ 10 4 cells/100 ⁇ L/well, and cultured in a 5% CO 2 incubator at 37° C. for 1 day or 2 days.
• SEQ-1 or any one of the nucleic acid complexes synthesized was added to reach a final concentration of 100 nmol/L, and incubation was performed in a 5% CO 2 incubator at 37° C. for 2 hours. Subsequent fluorescence imaging analysis was performed with the same method as in Test Example 1.
• Table 3 shows the results. It was revealed from the results that, as compared with SEQ-1, which contained no sugar ligand, nucleic acid complexes 1 to 5, which had mannose, were not effectively taken up by ASGR1-expressing cells, whereas nucleic acid complexes 6 to 8, which had GalNAc, were effectively taken up by ASGR1-expressing cells.
• the liver was perfused and liver parenchymal cells and Kupffer cells were separated through a flow cytometer, and fluorescence intensity was measured.
• the fluorescence intensity for the group with administration of SEQ-1 was defined as 1, and fluorescence intensity for each group with administration of any one of the nucleic acid complexes was calculated as a relative value.
• Table 4 shows the results. It was revealed from the results that, as compared with SEQ-1, which contained no sugar ligand, nucleic acid complex 3, which had mannose, was efficiently taken up by mouse Kupffer cells, which have been reported to be CD206-positive, and nucleic acid complex 6, which had GalNAc, was efficiently taken up by mouse liver parenchymal cells, which have been reported to be ASGR1-positive.
• Anhydrous zinc chloride (3.65 g, 26.8 mmol) was weighed in a 250-three-necked flask under argon. The flask was dried under reduced pressure at 110° C. for 1 hour., and then allowed to cool under reduced pressure overnight.
• the mixture was filtered through a 70-mL plastic phase separation cartridge, and treated with a solution of 20% piperidine in DMF (50 mL) for 30 minutes while bubbling with nitrogen was performed. The solution was discharged, and the resin was washed with DMF.
• the resin was transferred into a test tube of 25 ⁇ 150 mm, dried under reduced pressure for 30 minutes, then treated with DCM (5 mL) and TFA (5 mL), and shaken with a plate shaker for 2 hours. Subsequently, the mixture was filtered through a phase separation cartridge, and washed with DCM. The filtrate was concentrated under reduced pressure to afford compound 41 (0.5859 g).
• the mixture was heated in a nitrogen atmosphere at 70° C. (external temperature) for 3 hours.
• the mixture was allowed to cool, and diluted with ethyl acetate (10 mL)/saturated aqueous sodium hydrogen carbonate solution (5 mL).
• Zinc chloride (0.466 g, 3.42 mmol) put in a 50-mL three-necked flask was dried under reduced pressure at 110° C. for 95 minutes, and allowed to cool under reduced pressure overnight. The flask was purged with nitrogen, and a solution of compound 38 (1.00 g, 2.57 mmol) and compound 48b (0.97 g, 3.42 mmol) in DCE (10 mL) was added thereto. The mixture was heated in a nitrogen atmosphere at 70° C. (external temperature) for 3 hours. The mixture was allowed to cool, and diluted with ethyl acetate (20 mL)/saturated aqueous sodium hydrogen carbonate solution (10 mL).
• Zinc chloride (210 mg, 1.54 mmol) put in a 50-mL three-necked flask was dried under reduced pressure at 110° C. for 75 minutes, and allowed to cool under reduced pressure overnight. The flask was purged with nitrogen, and a solution of compound 38 (450.2 mg, 1.16 mmol) and compound 48c (503.4 mg, 1.54 mmol) in DCE (5 mL) was added thereto. The mixture was heated in an argon atmosphere at 70° C. (external temperature) for 3 hours. The mixture was allowed to cool, and diluted with ethyl acetate (10 mL)/saturated aqueous sodium hydrogen carbonate solution (5 mL).
• the molecular weights of the nucleic acid complexes synthesized in Examples shown here were determined by ESI-MS or MALDI-TOF-MS, Table 5 shows the sequences of the sense strands used, the sequence of the antisense strand, and their molecular weights.
• Table 6 shows the sequences and molecular weights of the nucleic acid complexes.
• si-RNAs targeting B2M were prepared.
• Table 7 shows the sense strands and antisense strands of the si-RNAs.
• the nucleic acid complexes (si-RNA) prepared in Examples 13 to 16 were each subcutaneously administered to BALB/c mice (three mice per group) at 1.0 mg/kg or 5.0 mg/kg, and the liver was collected 7 days after the administration.
• the MRNA expression levels of liver B2M and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal control were measured through qPCR with use of a TagMan Probe (Applied Biosystems).
• the B2M mRNA expression level in the mouse liver for the untreated group (Control) was defined as 100%, and the B2M mRNA expression levels (relative values) for the groups with administration of any one of the nucleic, acid complexes were calculated. Table 8 shows the results.
• siRNA-2, siRNA-3, siRNA-4, and siRNA-5 exhibited dose-dependent gene silencing effect. While almost no gene silencing effect was found for siRNA-1, which contained no sugar ligand, even at 5.0 mg/kg, the siRNAs containing a sugar ligand exhibited high gene silencing effect even at 1.0 mg/kg.
• Nucleic acid complexes were prepared with the same method as in Examples 9 to 11, except that Gapmer-type antisense having a 3-10-3 motif (ASO; ISIS-549148) was used as a nucleic acid and compound 44, 47, or 53a was used.
• Table 9 shows the sequence of the sense strand used, the sequence of the antisense strand, and their molecular weights.
• Table 10 shows the sequences and molecular weights of the nucleic acid complexes.
• nucleic acid complexes 17 to 19 were carried out in the same manner as in Test Example 1.
• Test Example 5 the fluorescence intensity in a well with addition of ASO_3′Cy3, which is a nucleic acid without modification with a sugar ligand, was defined as 1, and fluorescence intensity in each well with addition of any one of the nucleic acid complexes was calculated as a relative value.
• Table 11 shows the results. It was revealed from the results that, as compared with ASO_3′Cy3, which is a nucleic acid containing no sugar ligand, nucleic acid complexes 17 and 19, which had GalNAc, were not effectively taken up by CD206-expressing cells, whereas nucleic acid complex 18, which had mannose, was effectively taken up by CD206-expressing cells.
• nucleic acid complexes 17 to 19 were carried out in the same manner as in Test Example 2.
• Test Example 6 the fluorescence intensity in a well with addition of ASO_3′Cy3, which is a nucleic acid without modification with a sugar ligand, was defined as 1, and fluorescence intensity in each well with addition of any one of the nucleic acid complexes was calculated as a relative value.
• Table 12 shows the results. it was revealed from the results that, as compared with ASO_3′Cy3, which is a nucleic acid without modification with a sugar ligand, nucleic acid complex 18, which had mannose, was not effectively taken up by ASGR1-expressing cells, whereas nucleic acid complexes 17 and 19, which had GalNAc, were effectively taken up by ASGR1-expressing cells.

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